2011 - Rapid Simultaneous Determination of Telmisartan, Amlodipine Besylate and Hydrochlorothiazide in a Combined Poly Pill Dosage Form by Stability-Indicating Ultra Performance Liquid

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Research article Open Access

Rapid Simultaneous Determination of

Telmisartan, Amlodipine Besylate andHydrochlorothiazide in a Combined

Poly Pill Dosage Form by Stability-Indicating

Ultra Performance Liquid Chromatography

Santaji NALWADE * 1,2, Vangala RANGA REDDY 1,Dantu DURGA RAO

1, Inabathina KOTESWARA RAO

1

1 Analytical Research and Development, Integrated Product Development, Dr. Reddy’s Laboratories Ltd.,Bachupally, Hyderabad-500 072, India.2 Department of Chemistry, J.N.T. University, Kukatpally, Hyderabad-500 072, A.P., India.

* Corresponding author. E-mails: [email protected] or [email protected] (S. Nalwade)

Sci Pharm. 2011; 79: 69–84 doi:10.3797/scipharm.1006-10

Published: January 4th 2011 Received: June 24th 2010 Accepted: January 4th 2011

This article is available from: http://dx.doi.org/10.3797/scipharm.1006-10

© Nalwade et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproductionin any medium, provided the original work is properly cited.

Abstract

A simple, precise and rapid stability-indicating ultra-performance liquidchromatography (UPLC) method is developed for the simultaneous quantitativedetermination of Telmisartan, Amlodipine besylate and Hydrochlorothiazidefrom their innovative poly pill combination drug product in the presence ofdegradation products. It involves a 100 mm x 2.1 mm, 1.7 µm C-18 column. Theseparation is achieved on a simple gradient method. The mobile phase A

contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile inthe ratio 90:10, v/v , and mobile B contains a mixture of sodium perchloratebuffer pH 3.2 (0.053M): acetonitrile in the ratio 20:80, v/v . The flow rate is0.6 mL min−1 and the column temperature is maintained at 55°C.The gradientprogram (T/%B) is set as 0/5, 1.2/5, 1.6/40, 4/40, 4.1/5 and 4.5/5. The detectorwavelength is 271 nm for Hydrochlorothiazide and Telmisartan and 237 nm for Amlodipine. The retention times of Telmisartan, Amlodipine, and Hydrochloro-thiazide are 3.6 minutes, 3.2 minutes and 0.9 minutes; respectively. The totalruntime for the separation of the three active compounds and their degradationproducts is 4.5 minutes. The described method is validated with respect tosystem suitability, specificity, linearity, precision and accuracy. The precision of

the assay method is evaluated by carrying out six independent assays of T, A

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Sci Pharm. 2011; 79: 69–84

and H (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H). Theaccuracy of the method is evaluated in triplicate at three concentration levels,i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL−1 of T,0.08 mg mL−1 of A, 0.2 mg mL−1 of H). The described method is linear over the

range, 16 to 48 µg mL−1

for T, 2 to 6 µg mL−1

A and 5 to 15 µg mL−1

for H. Themethod is fast and suitable for high-throughput analysis allowing the analysis ofabout 250 samples per working day.

Keywords

Validation • Stability • UPLC • Simultaneous • Degradation • Method development

Introduction

Cardiovascular diseases (CVDs) are the disorders of heart and blood vessels andprimarily include coronary heart disease, hypertension, cerebrovascular disease,peripheral artery disease, rheumatic heart disease, congenital heart disease and heartfailure. CVDs are the major cause of death in developed countries and also are rapidlyemerging as a main cause of death in the developing world. An estimated 17.5 millionpeople died from CVDs till 2005, representing almost 30% of all the global deaths. It isprojected that almost 20 million people will die from CVDs by 2015. The major risk factorsinvolved in CVDs are high low density lipoprotein (LDL) cholesterol, raised blood pressure,increased serum homocysteine level and platelet aggregation, which are primarily causedby unhealthy diet, physical inactivity and tobacco use. A novel polypill formulation isdeveloped using drugs Telmisartan, Amlodipine besylate and Hydrochlorothiazide forCVDs.

Telmisartan (T) is an angiotensin II receptor (type AT1) antagonist used in themanagement of hypertension. Telmisartan prevents the constriction (narrowing) of bloodvessels (veins and arteries). Telmisartan is a non-peptide molecule and chemicallydescribed as potassium 4'-[(1,7'-dimethyl-2'-propyl-1H ,3'H -2,5'-bibenzimidazol-3'-yl)-methyl]biphenyl-2-carboxylate (Fig. 1).

Hydrochlorothiazide (H) is a thiazide diuretic (water pill) that decreases the amount of fluidin the body by increasing the amount of salt and water lost in the urine. Hydrochloro-thiazide is used to lower blood pressure and to decrease edema (swelling), it is chemically

described as 6-chloro-3,4-dihydro-2H -1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide(Fig. 1).

Amlodipine besylate (A) is in a class of drugs called beta-blockers. Beta-blockers affect theheart and circulatory system (arteries and veins). Amlodipine besylate is used to lowerblood pressure, lower heart rate, reduce chest pain (angina), and to reduce the risk ofrecurrent heart attacks. It is chemically described as 3-ethyl 5-methyl 2-[(2-amino-ethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylatebenzenesulfonate (Fig. 1).

The ever-increasing need for speed and efficient use of time in the pharmaceutical and

other fields, there is demand for the development of fast and high throughput analytical


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Sci Pharm. 2011; 79: 69–84

procedures. The rapid quantitative determination of combination drugs with big differencein label claims (40 mg for T and 5 mg for A) with shorter run times is a challenge. ForUPLC based assays, the processes of reducing analysis time while adequately resolvinganalytes from degradation products is often accomplished with column with small particles.

The theoretical advantages for small particles are to get well resolved peaks with hightheoretical plates over small concentration.

Present drug stability test guidance Q1A (R2) issued by international conference onharmonization (ICH) [1] suggest that stress studies should be carried out on a drugproduct to establish its inherent stability characteristics, leading to identification ofdegradation products and hence supporting the suitability of the proposed analyticalprocedures. It also requires that analytical test procedures for stability samples should bestability indicating and they should be fully validated.

Accordingly, the aim of the present study was to establish inherent stability of Telmisartan,

Amlodipine besylate, and Hydrochlorothiazide through stress studies under a variety ofICH recommended test conditions [1–3] and to develop a rapid stability-indicating reversephase assay method [4–6].

Literature survey reveals that a variety of spectrophotometric and chromatographicmethods including UV, colorimetric determination, ratio derivative, and a stability-indicating HPLC methods have been reported for determination T A and H either single orin combination with other drugs [7–12]. Whereas no liquid chromatography method hasbeen reported for simultaneous quantitative determination of T, A and H in the combineddosage form.

Hence a rapid simple reproducible Ultra performance liquid chromatography method wasdeveloped for simultaneous quantitative determination of T, A and H in polypill pharma-ceutical dosage forms in the presence of degradation products.

O

O

NH2O

O

O

NH

ClS

O

O

OH

SSNH

O O

NH

O

O

NH2

Cl

A TH

N

N

NN

O-

O K+

Fig. 1. Structure of Amlodipine besylate (A), Hydrochlorothiazide (H) andTelmisartan (T)


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Experimental

Chemicals and reagents

Standards and tablets (40 mg of Telmisartan, 5 mg of Amlodipine besylate, 12.5 mg of

Hydrochlorothiazide) were supplied by Dr. Reddy’s laboratories limited, Hyderabad, India.The HPLC grade acetonitrile and methanol, analytical grade sodium per chlorate anddisodium hydrogen phosphate were purchased from Merck, Darmstadt, Germany. Waterwas prepared by using Millipore MilliQ Plus water purification system

Instrumentation

Acquity UPLCTM system (Waters, Milford, USA) used consisting of a binary solventmanager, a sample manager and a UV detector. The out put signal was monitored andprocessed using empower software, water bath equipped with MV controller (Julabo,Seelbach, Germany) was used for hydrolysis studies. Photo stability studies were carriedout in a photo stability chamber (Sanyo, Leicestershire, UK). Thermal stability studies wereperformed in a dry air oven (MACK Pharmatech, Hyderabad, India).

Chromatographic conditions

The chromatographic column used was Acquity UPLC BEH shield RP-18, 100 mm x2.1 mm i.d with 1.7 µm particles. The mobile phase A contains a mixture of sodiumperchlorate buffer pH 3.2(0.053M): acetonitrile (90:10, v/v ) and mobile phase B contains amixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile (20:80, v/v ). The flowrate was 0.6 mL min−1 and column temperature was maintained at 55°C. The detectionwavelength was 271 nm for Hydrochlorothiazide and Telmisartan, and 237 nm for Amlodipine besylate. The diluent contains a mixture of sodium perchlorate buffer pH

3.2(0.053M): methanol in the ratio 90:10, v/v.

Preparation of standard solutions

Stock standard solutions of T, A and H (0.54 mg mL−1 of T, 0.2 mg mL−1 of A, 0.5 mg mL−1 of H) were prepared by dissolving an appropriate amounts of the compounds in methanol.Working solutions 0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H wereprepared from above stock solution in mobile phase for assay determination.

Preparation of sample solut ion

Weighed and crushed twenty tablets into a clean and dry mortar-pestle. An equivalent to

20 mg of H (64 mg of Telmisartan, 8 mg of Amlodipine besylate) was transferred to a 100mL volumetric flask, added 15 mL of 0.1% orthophosphoric acid and kept on rotary shakerfor 20 minutes. Added 50 mL of methanol and sonicated for 20 minutes. Made the volumeto 100 mL with methanol (0.64 mg mL−1 of T, 0.08 mg mL−1 of A, 0.2 mg mL−1 of H). About5 mL of supernant solution was taken and diluted to 100 mL with diluent to get workingconcentrators (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H). The solutionwas then filtered through 0.45 µ (Nylon 66- membrane) filter.


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Results and Discussion

Chromatographic Method Development

The method was optimized to separate major degradation products formed under different

stress conditions. The main target of the chromatographic method is to get the separationfor closely eluting degradation products, mainly the degradation product at 1.26 RRT (withrespect to A) that is eluting between A and T. The degradation samples were run usingdifferent stationary phases like C18, C8, and Mobile phases containing buffers likephosphate and perchlorate with different pH (2.5–7) and using organic modifiers likeacetonitrile and methanol in the mobile phase. The isocratic method was not working sincetelmisartan and amlodipine peaks were not separated with a proper resolution and also thedegradents of hydrochlorothiazide were not separating from three actives. Hence themethod is optimized with a gradient program. As the concentration of amlodipine is veryless as compared to telmisartan, so that to improve response, resolution and peak shapesthe method was tried at different column temperatures. But the separation, response of

peaks and peak shapes were satisfactory in the adopted chromatographic conditions only.It indicated that the gradient method with 10% acetonitrile as organic modifier in mobilephase A and 80% in mobile phase B was successful in separating drugs and allchromatographic degradation products. Some of the trials were summarized as below.

Selection of Diluent (Extracting solvent):

As the tablets are film coated,and all the active drugs are stable in acidic conditions, the0.1% orthophosphoric acid is added in order to open the film coating and to use the sameassay method for quantification of uniformity of dosage units by UPLC.The trials were 0.1N HCl also but the peak shapes are not good in 0.1N HCl. The chromatograph shown in

Fig.2a. As all the three actives T, A, & H are freely soluble in methanol, so it is used as theextracting solvent.

Fig. 2a. Chromatogram of T, A and H at 271 nm and 237 nm from drug product, as 0.1N HCl used in the sample preparation instead of 0.1 % OPA


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Selection of Stationary Phase:

The stationary phases like C8 and C18 were tried but on C8 the peak shape for T and thepeak symmetry factor for hydrochlorothiazide is more than 1.7, the separation between Aand T is not adequate as shown in Fig.2b. On the other hand the peak shapes for all thethree components are good and all the three peaks are well separated from each otherand their degradents. So that the column with C18 stationary phase is selected.

Fig. 2b. Chromatogram of T, A and H at 271 nm and 237 nm from drug product by using100 mm x 2.1 mm, 1.7 µm C-8 column with gradient program as 0/5, 1.0/5,1.2/40, 3/40, 3.3/5 and 4.0/5.

Selection of mobile phase buffer:

The buffers like Phosphate buffer With Buffer with pH 3.2, pH 7.5 and the perchloratebuffer with pH 2.5 and 3.2 were tried as mobile phase buffer. But with pH 2.5, pH 7.5phosphate and per chlorate buffer with pH 2.5 the results were not enough good in termsof peak separation and peak shapes, shown in Fig. 2c. On the other hand the perchloratebuffer with pH 3.2, the results were found satisfactory so that the mobile phase with pH 3.2perchlorate buffer is finalized.

Optimization of gradient program:

Before going to gradient program, an isocratic program with organic modifier methanol andacetonitrile were tried but all the three components are eluted at the same retention timewith both methanol and acetonitrile. As we are using the column temperature 55°C theacetonitrile is preferred as an organic modifier rather than methanol. The different gradientprograms tried are shown in the Fig. 2d.

Finalization of Chromatographic conditions

By considering all the experiments the chromatographic conditions are finalized. Thechromatograph with finalized chromatographic condition was shown in the Fig.3.


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Fig. 2c. Chromatograms of T, A, and H from drug product by using different mobilephase buffers.


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Fig. 2d. Chromatograms of T, A, and H from drug product by using different gradientprograms.

Further the analysis was performed for pharmaceutical dosage forms of both the labelclaim tablets (40 mg T, 5 mg A, 12.5 mg H and 20 mg T, 2.5 mg A, 6.25 mg H). The assay(% of active drug content present in the tablets with respect to its label claims) results fordrug product, 99.8%, 99.5%,of T, 99.7%, 98.8% of A and 100.1%, 99.8% of H,respectively.

Analysis also performed (n = 3) separately for Telmisartan, Telmisartan and Hydrochloro-thiazide tablets and Amlodipine besylate and Hydrochlorothiazide tablets. The assayresults for Telmisartan were 99.5%, 99.7%, 99.9%. The assay results for Telmisartan andHydrochlorothiazide were, 99.7%, 99.5% and 99.9% for Telmisartan and 99.3%, 98.5%and 99.1% for Hydrochlorothiazide. The assay results for Amlodipine besylate andHydrochlorothiazide were, 99.5%, 99.7% and 98.7% for Amlodipine besylate and 99.8%,98.5% and 99.3% for Hydrochlorothiazide. It indicated that, adopted UPLC method alsocan be used separately for assay estimation of Telmisartan tablets, simultaneousestimation of Telmisartan and Hydrochlorothiazide tablets and simultaneous estimation of Amlodipine besylate and Hydrochlorothiazide tablet.


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Fig. 3. Typical chromatogram of Telmisartan, Amlodipine besylate andHydrochlorothiazide at 271 nm and 237 nm from drug product.

Method validation

System suitability

In order to find the adequate peak separation (resolution) and repeatability of the proposedmethod, suitability parameters including retention factor, selectivity and asymmetry factorwere investigated and the results were abridged in Table 1.

Specificity

The specificity of an analytical method is the ability of the method to determine the analyteresponse in the presence of additional components such as impurities, degradationproducts and matrix [4]. The solution of analytical placebo (containing all the excipientsexcept T, A, & H) was prepared according to the sample preparation procedure andinjected. To identify the interference by these excipients, a mixture of inactive ingredients,

standard solutions, and the commercial pharmaceutical preparations including T, A, and Hwere analyzed by the developed method.

The specificity of the method was also evaluated to ensure there were no interferenceproducts resulting from forced degradation.

Placebo Interference

The placebo (excepients present in the tablet) sample were prepared as per the testmethod and analyzed in the UPLC. The Fig.4. shows there is no peaks at the retentiontime of the T, A, & H in the chromatograph indicates that there is no placebo interference.


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Fig. 4. Typical chromatogram of Telmisartan, Amlodipine besylate and Hydrochloro-thiazide tablets placebo blend at 271 nm and 237 nm from drug product

Forced degradation studies.

Stress testing of a drug substance can help to identify the likely degradation products,which can help to establish the degradation pathways and the intrinsic stability of themolecule.

All stress decomposition studies were performed at an initial drug concentration 0.64 mgmL−1 of T, 0.08 mg mL−1 of A, 0.2 mg mL−1 of H. The degradation conditions are selectedon the basis of literature survey [13-17].

Water induced degradation

Water hydrolysis was performed in purified at 60 °C for 6 hours. Fig. 5a shows the majordegradation found at RRT 0.83 with respect to H. and all the major and minor degradationproducts were well separated from T, A, and H peaks. The peak purity is checked for T, A,and H and the results are summarized in Table 2.

Hydrogen peroxide induced degradation

For hydrogen peroxide-induced degradation, the studies were carried out at roomtemperature in 1% hydrogen peroxide for 6 hours. The Fig.5a. shows the majordegradation found at RRT 0.841 with respect to H and at RRT 1.056 and 1.272 withrespect to A. All the major and minor degradation products were well separated from T, A,and H peaks. The peak purity is checked for T, A, and H and the results are summarizedin Table 2.

Acid induced degradation

Acid hydrolysis was performed in 1N HCl at 60 °C for 6 hours. Fig. 5a shows the major

degradation found at RRT 0.842 with respect to H. and all the major and minor


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degradation products were well separated from T, A, and H peaks. The peak purity ischecked for T, A, and H and the results are summarized in Table 2.

Base induced degradation

The study in basic solution was carried out in 0.1N NaOH at 60 °C for 6 hours. The Fig.5b. shows all the three T, A, and H were stable in 0.1N NaOH and all the minordegradation products were well separated from T, A, and H peaks. The peak purity ischecked for T, A, and H and the results are summarized in Table 2

Fig. 5a. Typical chromatograms of Telmisartan, Amlodipine besylate and Hydrochloro-thiazide at 271 nm and 237 nm for force degradation study

Heat induced degradation

The drug product was exposed to dry heat at 105 °C for 24 hours. Samples werewithdrawn at appropriate time and subjected to UPLC analysis after suitable dilution (0.032mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H).The Fig. 5b. shows that the drugproduct containing T, A, and H is thermally stable. The peak purity is checked for T, A, and

H and the results are summarized in Table 2.


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Photo-degradation

Photo degradation studies were carried out at according to option 2 of Q1B in ICHguidelines [3]. Samples were exposed to light for an overall illumination of 1.2 million luxhours and an integrated near ultraviolet energy of 200 watt hm2. Samples were withdrawnat appropriate time and subjected to UPLC analysis after suitable dilution (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H). The drugs T, A and H are stable underphotolytic condition. The peak purity is checked for T, A, and H and the results aresummarized in Table 2.

Fig. 5b. Typical chromatograms of Telmisartan, Amlodipine besylate and Hydrochloro-thiazide at 271 nm and 237 nm for force degradation study

Linearity

Linearity solutions were prepared from stock solution at six concentration levels from 50 to150% of analyte concentrations (16 to 48 µg mL−1 for T, 2 to 6 µg mL−1 A and 5 to 15 µgmL−1 for H). The linear regression analysis of T, A, and H were constructed by plotting thepeak area of the analytes (y) versus analytes concentration in (x) axis. The calibrationcurves were linear in the range of 16 to 48 µg mL−1 for T, 2 to 6 µg mL−1 A and 5 to 15 µgmL−1 for H with a correlation coefficient of more than 0.999 for all the three drugs. Theslope, Y-intercept and correlation coefficient were calculated and summarized in Table 3.


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Precision

The precision of the assay method was evaluated by carrying out six independent assaysof T, A and H (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H) test samplesagainst qualified reference standard. The percentage of RSD of six assay values wascalculated. Different analyst from the same laboratory evaluated the intermediate precisionof the method. The R.S.D. values of intra- and inter-day studies for T, A, and H confirminggood precision of the method (Table 4).

Tab.1. System suitability parameters for T, A and H

System suitability test parameters T A HRetention time(min)(mean ± S.D., n = 5)

3.944± 0.0018

3.332± 0.0010

0.913± 0.001

Repeatability of retention time;R.S.D. % (n = 5)

0.0460 0.0300 0.3342

Repeatability of peak area;R.S.D.% =(S.D./mean) x 100

0.1501 0.5403 0.0752

Resolution (Rs) 8.3 36.9 –Tailing factor (asymmetric factor) 1.2 1.2 1.4Retention factor(k’) 2.944 2.332 0.082USP plate count 34765 43314 2974

Tab. 2. Peak purity results of T, A and H

Purity Angle Purity Threshold Purity FlagStress ConditionT A H T A H T A H

Heat Stress 0.070 1.118 0.114 0.538 4.580 1.140 No No No Aqueous Stress 0.057 0.902 0.131 0.499 3.973 1.384 No No No Acid Stress 0.064 1.108 0.108 0.493 4.301 1.125 No No NoBase Stress 0.065 1.306 0.122 0.536 5.057 1.212 No No NoPeroxide Stress 0.060 1.727 0.241 0.533 5.776 1.202 No No No

Tab. 3. Linearity

AnalyteConcentration

ranger a Slope intercept

95% confidenceinterval forintercept

Potentialassaybias

T16.024–48.073

µg.ml−1 0.9999 6282.0 −888.486

877.9552 &−2656.9267

−0.4

A2.020–6.020

µg.ml−1 0.9996 10044.1 −14.5193

596.3377 &−625.3762

0.0

H5.000–15.000

µg.ml−1 0.9998 13499.7 −827.248

2710.7834 &−4365.2787

−0.6a Correlation coefficient.


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Accuracy

The accuracy of an analytical method expresses the nearness between the referencevalue and found value. The accuracy of the method was evaluated in triplicate at threeconcentration levels, i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL−1 of T, 0.08 mg mL−1 of A, 0.2 mg mL−1 of H) in tablets. The results obtained are shown inTable 5.

Tab. 4. Intra-day and inter-day Precision results of T, A and H from tablets (n = 6)

Pre-1 Pre-2 Pre-3 Pre-4 Pre-5 Pre-6ActiveName % Assay

% RSD % Mean

T 100.2 99.7 100.2 100.4 99.4 100.6 0.45 100.1 A 99.0 99.3 98.6 99.4 101.2 99.2 0.90 99.5

Intra-dayprecision

H 98.9 98.8 99.3 99.9 100.1 99.8 0.54 99.5T 98.0 100.0 100.9 99.0 99.3 100.4 1.04 99.6

A 99.9 99.4 98.4 99.4 99.8 99.4 0.53 99.4Inter-dayprecision

H 100.0 100.6 100.6 99.7 100.0 99.3 0.51 100.0

Tab. 5. Accuracy Results of T, A and H from tablets (n = 6)

AnalyteRecovery

level

ActualConc.

(µg.ml−1)

Found Conc.(µg.ml−

1)

% Recovery % R.S.D. % Error a

50% 320 321.86±0.567 100.58 0.564 0.581

100% 640 640.51±0.449 100.08 0.449 0.080T150% 960 964.51±0.638 100.47 0.635 0.470

50% 40 39.99±0.480 99.97 0.480 −0.030100% 80 79.56±0.903 99.45 0.908 −0.550 A150% 120 119.78±0.194 99.82 0.194 −0.183

50% 100 993.2±0.492 99.32 0.495 −0.683100% 200 198.94±0.547 99.47 0.549 −0.530H150% 300 298.50±0.297 99.50 0.298 −0.750

a [found conc. – actual conc./actual conc.] x 100.

Solution Stability and Mobile Phase Stability

The solution stability of T, A and H was carried out by leaving the test solution in tightlycapped volumetric flask at room temperature for 48 hrs. The same sample solution wasassayed for a 24 hours interval up to the study period against freshly prepared standardsolution of T, A and H. The mobile phase stability was also carried out by assaying thefreshly prepared standard solution for 24 hours interval up to 48 hours. The mobile phasepreparation was kept constant during the study period. The percentage RSD of assay of T, A and H was calculated for the study period during mobile phase and solution stabilityexperiments. The % RSD of the assay of T, A, and H during solution stability and mobilephase experiments were within 1% and it indicates that both standard and test preparation

and mobile phase were stable for 2 days on bench top at room temperature.


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Conclusions

The established UPLC method proves to be simple, linear, precise, accurate and specific.The total runtime was 4.5 minutes within which three drugs and their degradation productswere separated. The method was validated and shows satisfactory data for all the methodvalidation parameters tested. The Developed method is stability indicating and can beused for simultaneous quantitative determination of the drugs T, A and H in presence ofdegradation products in stability by the industry. The adopted UPLC method also can beused separately for assay estimation of Telmisartan tablets, simultaneous estimation ofTelmisartan and Hydrochlorothiazide tablets and simultaneous estimation of Amlodipinebesylate and Hydrochlorothiazide tablets.

Acknowledgement

The authors wish to thank the management of Dr. Reddy’s Laboratories Ltd. for supporting

this work. Cooperation from colleagues and of Research & Development and AnalyticalResearch & Development of Dr. Reddy’s Laboratories Ltd. is appreciated.

Dr. Reddy’s internal publication number for this manuscript is PUB00092-10.

Authors’ Statement

Competing Interests

The authors declare no conflict of interest.

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2011 - Rapid Simultaneous Determination of Telmisartan, Amlodipine Besylate and Hydrochlorothiazide in a Combined Poly Pill Dosage Form by Stability-Indicating Ultra Performance Liquid